SE545539C2 - A method for multiplexed detection of one or more target biomolecules in situ - Google Patents

Abstract

The disclosure relates to a method for multiplexed detection of one or more target biomolecule(s) in situ using optical encoding, wherein each target biomolecule has at least one detection target, comprising the steps of: a. providing one or more nanoparticle type(s) in a suspension, each nanoparticle having a coating that provides binding affinity of the nanoparticle to a type-specific detection target, and wherein each nanoparticle comprises one or more fluorophore(s) that generates a signal which is unique for each nanoparticle type; b. providing one or more cells to be penetrated by the nanoparticle(s), wherein the cell(s) comprise(s) one or more target biomolecule(s); c. optionally preparing the target biomolecule(s) for binding with the nanoparticle(s), such as binding the target biomolecule(s) with at least one molecule comprising the at least one detection target, and/or amplifying the detection target in situ; d. contacting the cells with the suspension comprising the nanoparticle(s), thereby allowing the nanoparticle(s) to penetrate the cells in order to bind with the detection target of the target biomolecule; e. optically decoding the fluorophore signal(s) emitted by the nanoparticle of the nanoparticle type hybridized to the detection target of the target biomolecule(s) by measuring the wavelength and intensity of the emitted signal(s), thereby detecting the presence and identity of the target biomolecule(s).

Description

A method for multiplexed detection of one or more target biomolecules in situ Technical field The present disclosure relates to a method for multiplexed detection of one or more target biomolecules in situ. More specifically, the disclosure relates to a method for multiplexed detection of one or more target biomolecule in situ as defined in the introductory parts of claim 1, and a kit of parts as defined in claim Background art Many bioanalytical technologies typically use fluorescent methods to allow for multiplexed (more than one) detection of various targets. Several applications benefit from doing this such as histology, flow cytometry, fundamental cellular and molecular protocols, fluorescence in situ hybridization, DNA sequencing, immuno assays, binding assays etc. While it is possible to detect more than one target simultaneously, using fluorophores still severly limits the ability to multiplex. For the in situ hybridization application especially new technologies are emergin in order to increase the magnitude of multiplexing capacity focused on barcode decoding via in situ sequencing schemes.

The use of multiple fluorophores (dyes) is limited by its physical properties, where each dye has a certain spectral shape of its absorbance and emission (about 100 nm broad for organic dyes). Due to this, when multiple fluorophores are used simultaneously their emission needs to be well separated. This causes a limitation in the amount of dyes that can be used simultaneously before the emission overlaps (spectral overlap) such that it becomes difficult to distinguish one dye from another (typically less than 5-10 dyes simultaneously). ln order to overcome this, in situ sequencing technologies are emerging (Strell et al. (FEBS J. (2018), p. 14435 (Placing RNA in context and space-methods for spatially resolved transcriptomics)) and Lein et al. (Science (2017) 358,64-69 (The promise of spatial transcriptomics for neuroscience in the era of molecular cell typing.)) are two relevant articles in this context). These make use of molecular DNA barcodes that represent each a unique target that needs to be decoded for the successful detection to be completed (targeted method), or alternatively doing the direct sequencing of the target in case of RNA/DNA (non-targeted). The process of decoding involves the use of DNA sequencing Chemistry (addition of signal and removal), the iterative process of stepping one DNA base at a time and repeating this the amount of times necessary depending on the level of multiplexing desired, maintaining the integrity and location of sample during this iterative process, image acquisition between each iteration, and finally the image analysis to puzzle and align all images together to thereafter decide on the identity of each target. The whole process therefore takes much longer time than that of one-step (direct) detection methods, and typically requires trained personnel and/or robust hardware that integrates chemical steps with imaging steps resulting in high costs. Furthermore, the process of doing several steps iteratively also makes the sequencing approach sensitive to errors or mistakes, as it is enough for a sub-part of the process to go wrong for the whole decoding to fail altogether. The sequencing process is therefore more complicated, sensitive to errors, expensive, and time consuming in comparison to a direct approach.

There is thus a need for improved methods for multiplexed detection of one or more target biomolecules, taking into account the problems of the existing solutions.

Summary of the invention lt is an object of the present disclosure to mitigate, alleviate or eliminate one or more of the above-identified deficiencies and disadvantages in the prior art and solve at least the above mentioned problem.

According to a first aspect there is provided a method for multiplexed detection of one or more target biomolecule in situ using optical encoding, wherein each target biomolecule has at least one detection target, comprising the steps of: a. providing one or more nanoparticle type(s) in a suspension, each nanoparticle having a coating that provides binding affinity of the nanoparticle to a type-specific detection target, and wherein each nanoparticle comprises one or more fluorophore(s) that generates a signal which is unique for each nanoparticle type; b. providing one or more cells to be penetrated by the nanoparticle(s), wherein the cell(s) comprise(s) one or more target biomolecule(s); c. optionally preparing the target biomolecule(s) for binding with the nanoparticle(s), such as binding the target biomolecule(s) with at least one molecule comprising the at least one detection target, and/or amplifying the detection target(s) in situ; d. contacting the cells with the suspension comprising the 3 nanopartic|e(s), thereby allowing the nanopartic|e(s) to penetrate the cells in order to bind with the detection target(s) of the target biomolecule; e. optically decoding the fluorophore signal(s) emitted by the nanoparticle of the nanoparticle type bound to the detection target of the target biomolecule(s) by measuring the wavelength and intensity of the emitted signal, thereby detecting the presence and identity of the target biomolecule(s).

Hereby, a method of simultaneously detecting a significant plurality (50-100) of detection targets in situ using optically encoded nanoparticles in a one-step direct manner is provided. This can be achieved by tailoring the nanoparticles to achieve distinct and precise incorporation of fluorophores in well-defined nanoparticles and thereby optically encoding such. The nanoparticles can in addition provide protection for the fluorophores to prevent bleaching. This is important because the number of combinations becomes limited if the intensity distributions overlap with each other. Furthermore, it is possible to utilize semiconductor fluorophores such as quantum dots to achieve a greater range of combinations, either by incorporating multiple such or in combination with organic fluorophores. The number of combinations becomes nm-l, where n is the number of intensity levels and m the number of colors. lt follows that the number of combinations scales greater with the number of colors rather than the number of intensity levels. Using semiconductor fluorophores it is possible to achieve both a narrower emission spectrum (enabling more colors in parallel before spectral overlap becomes a problem, as well as fitting in more colors in a spectrum together with organic fluorophores due to the possibility of having semiconductor fluorophores with very large stoke shifts. By using nanoparticles it is also possible to alter the properties of individual fluorophores when incorporated in an organized matter in a particle where distance between the fluorophores can be carefully controlled, such that they can be coupled energetically and act as waveguides/antennas, such as Förster resonance energy transfer (FRET) or excitation energy transfer (EET). This allows for further flexibility in achieving a higher number of concentration levels and/or higher number of color combinations, leading ultimately to a higher number of multiplexity.

The invention allows for the process of nanoparticles with a certain unique identity binding to a detection target (DNA/RNA/protein) in situ, where the size of the nanoparticles allows for the penetration of cells and that the nanoparticles can immobilize onto the target while the excess is washed away. Preferably, the detection target is designed such that a plurality ofnanoparticles can attach within at least one optically resolvable pixel forming a cluster of nanoparticles in the spot of detection target, such that the signal intensity becomes higher in this cluster compared to the signal from single nanoparticles that may bind non-specifically to the surrounding, allowing for a localized signal to be detected upon target binding, where the unique identity of the nanoparticle encodes for the target identity. Typically this may be done by performing target amplification prior to decoding, such as rolling-circle amplification (RCA) or other means of localized/clonal amplification (hairpin chain reaction etc.), or by designing probes in a manner similar to traditional fluorescent in situ hybridization (FISH) techniques such that they can form a localized cluster.

According to some embodiments, each nanoparticle type is optically encoded by (i) incorporating precisely controlled ratios of at least one fluorophore, thereby controlling the emission wavelength and intensity from the nanoparticle type, or (ii) altering the properties of the fluorophore(s) affecting its emission intensity.

Thus, the invention allows for the use of optically encoded nanoparticles that have been synthesized with the incorporation of precise controlled intensity levels of dyes such that each the combination of levels (ratios) represents a unique identity, enabling higher number of multiple identities compared to single colors alone. The number of combinations becomes nm- 1, where n is the number of intensity levels and m the number of colors.

According to some embodiments, the binding affinity of the coating is provided by a detection probe X attached via a linker to the nanoparticle, wherein detection probe X is chosen from a nucleic acid molecule, an antigen or an antibody.

This design allows for rapid and custom modification ofthe nanoparticles with various targets to easily create new panels as wished for by the end user.

According to some embodiments, the coating furthermore comprises a repulsive component provided by a functional group Y attached via a linker to the nanoparticle, wherein functional group Y is chosen from a charged group with a positive or negative charge, a zwitterionic group the method comprises both a positive or negative charge, or a sterically repulsive functional group such as polymer chain or an aliphatic chain.Thus, the carefully tailored surface coating of the nanoparticles may comprise a repulsive part and a attractive part (with detection probe) such that they repulse each other and other surfaces to form a stable dispersion while still being able to form specific attractive bonds with a detection target (DNA/RNA or antibodies) resulting in that the nanoparticles attach and immobilize to the detection target. Efficient attachment happens when the repulsive forces are balanced with the attractive forces of the probe.

According to some embodiments, the linker comprises an anchor group which tethers the coating to the nanoparticle and optionally a spacer group.

An anchor group "tethering" the coating to the nanoparticle is to be interpreted as that the anchor group binds the coating to the nanoparticle by covalent or non-covalent binding.

According to some embodiments, one or more linkers can provide one or more detection probes X and/or functional groups Y, or where multiple linkers can facilitate provide multiple detection probes and/or functional groups Y via an interconnecting backbone.

Hereby, a plurality of combinations of linkers and detection probes X and/or functional groups Y may be used.

According to some embodiments, the one or more fluorophores are selected from organic fluorophores, chosen from Atto 425, Cy3, Cy5 and Cy7, or from different colors of inorganic fluorophores, wherein the inorganic fluorophores comprises quantum dots, rods, perovskite quantum dots or metal-ligand complexes.

By using well separated fluorophores with high photostability it is possible to achieve a high quality incorporation ofthese fluorophores in the nanoparticle while minimizing spectral overlap. Both factors are important in order to achieve well separated intensity distributions acting as unique ba rcode identities for each batch of nanoparticles (see figure 4,5,6).

According to some embodiments, the one or more fluorophores are selected from a combination of different colors of organic fluorophores and different colors of inorganic fluorophores, wherein the inorganic fluorophores comprises quantum dots, rods or perovskite quantum dots.By using nanoparticles as probes the possibility to combine a broader selection of fluorophores is permitted, especially for the case of semiconducting fluorophores such as quantum dots as mentioned above. This is important because the number of possible combinations scales greater with the number of colors used compared to the number of levels (distinguishable dye concentrations) with the formula m", where m is the number of intensity levels and n is the number of colors. Using semiconductor fluorophores it is possible to achieve both a narrower emission spectrum (enabling more colors in parallel before spectral overlap becomes a problem, as well as fitting in more colors in a spectrum together with organic fluorophores due to the possibility of having semiconductor fluorophores with very large stoke shifts.

According to some embodiments, the nanoparticle has at least one of the following characteristics: i. it is a spherical particle comprising of silica and/or semiconductor, organic, inorganic, metal and/or polymer material; ii. it has a diameter of less than 300 nm, preferably less than 200 nm, and more preferably less than 100 nm, even more preferably less thanHm.

Spherical silica particles are known from the field, and are an alternative for use in the present invention. Also, nanoparticles of polymers and/or plastics may be used.

Using a particle with a size smaller than about 300 nm, and preferably about 100 nm ensures for penetration into cells and that the method can be performed in situ. For the particle to bind to the detection target (after having entered the cell), a smaller size is typically adavantageous.

According to some embodiments, in step c, the target biomolecule is prepared by binding to it at least one molecule the method comprises the detection target, such as a barcoded nucleic acid, padlock probe or initiator sequence for subsequent amplification.

This enables the NP probes to be used in assays where enzymatic amplification is omitted, such as regular in situ hybridization (ISH) methods where at least one, but preferably multiple detection targets are bound to a biomolecule in order to generate a stronger signal by binding multiple NPs to a biomolecule. 7 According to some embodiments, the detection target comprises a nucleic acid molecule, which is, or facilitates a molecule that is, amplified using RCA or multiple hybridization events.

Hence, the signal detection becomes easier and robust due to (i) a higher signal intensity (ii) the necessity of multiple binding events to generate said signal to avoid randomly immobilized nanoparticles and (iii) utilizing the specificity of molecular tools such as padlock probes for the amplification ofthe target, avoiding non-targets to be amplified and therefore detected. Similarly, other amplification methods utilizing the binding of for example two or more detection targets in close proximity to allow subsequent hybridizations to generate a stronger but specific signal can be achieved.

According to some embodiments, the decoding is effected by optical decoding such as by optical imaging.

This enables high resolution spatial information to be collected with the throughput and resolution of the imaging system and can enable large areas to be scanned.

According to some embodiments, the target biomolecule is further co-labelled with small molecule dyes/probes together with the nanoparticles.

According to some embodiments, further providing one or more molecular probes, each molecular probe comprises a fluorophore that is bound to a nucleic acid molecule, an antigen or an antibody providing binding affinity of the molecular probe to the specific detection ta rget.

This further improves both robustness and multiplexing where signal detection will rely on both nanoparticle binding event as well as molecular probe binding events to ensure that non- specifically bound nanoparticles are filtered from data analysis. ln addition, this can further be utilized to increase the multiplexing capacity by introducing different color of fluorophores acting as molecular probes.

According to a second aspect, the invention relates to a kit of parts, comprising, in separate containers: (i) nanoparticle(s) of one or more types in a suspension, each nanoparticle type having a coating that provides binding affinity of the nanoparticle to a specific detection 8 target, and wherein each nanoparticle comprises one or more fluorophores that generates a signal which is unique for each nanoparticle type, optionally nanoparticle(s) of each type in a suspensions, each nanoparticle type having a coating without binding affinity of the nanoparticle to a specific detection target wherein the binding affinity is provided in a subsequent step by a detection probe X attached via a linker to the nanoparticle, wherein the detection probe X is chosen from a nucleic acid molecule, an antigen or an antibody; (ii) optionally, ingredients for providing the one or more nanoparticle type(s) with binding affinity to a specific detection target, comprising a reaction buffer facilitating the binding of detection probe X to the linker, a washing buffer, and a suspension buffer to suspend the nanoparticles in after the introduction of the binding affinity to the coating; (iii) a probing buffer, comprising a solution with controlled pH, salt concentration and additives facilitating specific detection target binding of the nanoparticle(s); and (iv) instructions for use of the kit in the method according to the first aspect of the invention.

Thus, among the advantages and unexpected effects ofthe present invention, the following may also be disclosed: 0 Time can be saved, experimentally and imaging wise, due to one step vs. multiple for sequencing. 0 Costs can be saved due to fewer steps. 0 The method ofthe present invention is less sensitive to handling errors such as sample integrity and other risks of failure during sequencing procedures. 0 The present invention allows for an easier data analysis due to one set of images instead of multiple which requires aligning. 0 The present invention can be used with simple hardware setup by a regurarly trained lab person. 0 The present invention does not require fluidic solutions for iterative chemistry/imaging steps. 9 0 The present invention may lower the barrier for adoption of in-situ methods in regular laboratories that lack advanced facilities and instrumentation. 0 The present invention may be used for multiple applications where a localized signal needs detection/identification.

The present disclosure will become apparent from the detailed description given below. The detailed description and specific examples disclose preferred embodiments ofthe disclosure by way of illustration only. Those skilled in the art understand from guidance in the detailed description that changes and modifications may be made within the scope ofthe disclosure.

Hence, it is to be understood that the herein disclosed disclosure is not limited to the particular component parts of the device described or steps of the methods described since such device and method may vary. lt is also to be understood that the terminology used herein is for purpose of describing particular embodiments only, and is not intended to be limiting. lt should be noted that, as used in the specification and the appended claim, the articles "a", "an", "the", and "said" are intended to mean that there are one or more ofthe elements unless the context explicitly dictates otherwise. Thus, for example, reference to "a unit" or "the unit" may include several devices, and the like. Furthermore, the words "comprising", "including", "containing" and similar wordings does not exclude other elements or steps.

Brief description of the drawings The above objects, as well as additional objects, features and advantages of the present disclosure, will be more fully appreciated by reference to the following illustrative and non- limiting detailed description of example embodiments of the present disclosure, when taken in conjunction with the accompanying drawings.

Figure 1 shows the principle of the invention, wherein (a) shows the situation where one or more nanoparticles (NP) has binding affinity to bind to a specific detection target in a target biomolecule (rolling circle product (RCP)), and (b) shows the situation where the nanoparticle lack binding affinity to the target biomolecule (i.e. no detection target specific for the nanoparticle is present in the biomolecule).

Figure 2 shows a principal scheme for the preparation ofthe target biomolecule of the present invention ("Target preparation |P").

Figure 3 shows the principles for the coating of the nanoparticles in accordance with the method of the invention ("Coating details for |P").

Figure 4 shows the principle of optical encoding/decoding with three nanoparticle types with each a unique optical encoding based on ratiometric control of wavelength and intensity. Spheres represent nanoparticle type with fluorophore(s) incorporated denoted A-C inside the sphere. Coating is established on the nanoparticle surface wherein binding affinity is provided by the detection probe A-C. Decoding is effected by measuring the wavelength and intensity emitted by the nanoparticles, and a ratiometric decoding is illustrated for nanoparticle type A,B and C respectively (1:3:3), (1:0:2) and (3:3:O).

Figure 5 shows the overlaid emission spectrum of three wavelengths (fluorophores) and three intensity levels.

Figure 6 is a simulated ratiometric XY-plot of intensity distributions with size and intensity distribution imperfections for a multiplexing system with a population of n=30 with three wavelengths (fluorophores Cy3/Cy5/Cy7) and three intensity levels (relative 0-1-2) Figure 7 is a simulated 3D-plot showing the average ratiometric values of each nanoparticle type population for a 26-plex system with three wavelengths (fluorophores Cy3/Cy5/Cy7) and three intensity levels (relative 0-1-2) Figure 8 discloses fluorescence intensities measured for 11 different NP type batches produced with precise control of dye incorporation showing the realization of optical encoding of the na nopa rticles.

Figure 9 discloses NP binding (A) specifically to complementary detection targets on biomolecules (RCPs) and (B) not binding to non-complementary targets showing the specificity of the probing properties ofthe nanoparticle coating.

Figure 10 (A), shows 2-color encoded NP bound to biomolecules (RCPs) together with a molecular probe. (B), A line profile shows the signal emitted in respective channels (Cy3 & Atto 425 from NP and Cy5 from molecular probe). (C) Shows the respective channels for one RCP 11 imaged. (D), shows an XY-plot of the intensity distribution of the decoded NP signal from several RCPs signifying a particular nanoparticle type.

Figure 11 shows binding of NPs in cells to amplified biomolecules (RCPs) showing the action of in-situ detection of biomolecules using NPs.

The term "binding" with a target biomolecule, a nanoparticle and/or a detection target is to be interpreted as including the alternative of "hybridization" of nucleic acid molecules to each other.

The term "optical encoding" is to be interpreted as giving a particle a unique signal by incorporating a combination of multiple wavelengths and intensities of fluorophores The term "nanoparticle type" is to be interpreted as a nanoparticle having a coating that provides binding affinity to a specific detection target and a unique optical encoding. One or more nanoparticle types may be used in accordance with the invention, the various types having binding affinity for different targets. Each nanoparticle type will be represented by a plurality of nanoparticles having the same coating and optical encoding.

Detailed description The present disclosure will now be described in further detail. The disclosure may, however, be embodied in other forms and should not be construed as limited to the herein disclosed embodiments. The disclosed embodiments are provided to fully convey the scope ofthe disclosure to the skilled person.

The first aspect of this disclosure shows a method for multiplexed detection of one or more target biomolecules in situ, wherein each target biomolecule has at least one detection target, using optical encoding, comprising the steps of: a. providing one or more nanoparticles in a suspension, each nanoparticle having a coating that provides binding affinity of the nanoparticle to a specific detection target, and wherein each nanoparticle comprises one or more fluorophores that generates a signal which is unique for each nanoparticle; b. providing one or more cells to be penetrated by the nanoparticles, wherein the cells comprises one or more target biomolecules; c. optionally preparing the target biomolecules for binding with thenanoparticles, such as binding the target biomolecules with a molecule comprising the detection target, and/or amplifying the detection target in situ; d. contacting the cells with the suspension comprising the nanoparticles, thereby allowing the nanoparticles to penetrate the cells in order to bind with the detection target of the target biomolecule; e. optically decoding the fluorophore signals emitted by the fluorophore of the nanoparticle hybridized to the detection target of the target biomolecules by measuring the wavelength and intensity of the emitted signals, thereby detecting the presence ofthe target biomolecules.

Figure 1 shows the principles ofthe present method. Figure 2 shows the principles for preparing a target for multiplexed detection. ef; Figure 3 shows some alternatives for the coating of the nanoparticles of the invention.

The binding affinity of the coating may be provided by a detection probe X attached via a linker to the nanoparticle, wherein detection probe X is chosen from a nucleic acid molecule, an antigen or an antibody.

The coating may furthermore comprise a repulsive component provided by a functional group Y attached via a linker to the nanoparticle, wherein functional group Y is chosen from a charged group with a positive or negative charge, a zwitterionic group the method comprises both a positive or negative charge, or a sterically repulsive functional group such as polymer chain or an aliphatic chain.

Also, the linker may comprise an anchor group which tethers the coating to the nanoparticle and optionally a spacer group.

See table 1 for examples of anchor groups and functional groups Y, depending on the surfaces of the nanoparticle. See table 2 for examples of spacer groups.

Surface Anchor & Functional groups Compatible Anchor & Functional groupsMetal (Au) Thioi, Disuifide Carboxyiic acid :äminæ Anfimøniaim catšcan Phøsphønšc acid Sšiane Qrgariøsíiane Saiiføriate Phøsphšfie Hydroxyi Catechol Gallol Silica Ethoxysilane Methoxysilane Silazane Chlorosilane Functional group mediated Amine lsothiocyanate lsocyanate Sulfonyl Chloride Aldehyde Carbodiimide Acyl azide Anhydflde Fluorobenzen Carbonate NHS ester lmidoester Eoixude Fluoropheyl ester PhosphineCarboxylic acid Thiol Maleimide Haloacetyl (Br-/I-) Pyridyl disulfide Thiosulfonate Vinylsulfone Aldehylde Alkoxyamine Hydrazide Click Azide Alkyne Cyclooctines (Dibenzocyclooctyne (DBCO), trans-cyclooctene (TCO)) Cyclononyne (bicyc|o[6.1.0]nonyne (BCN)) Tetrazine Ligand Avidin Streptavidin NeutrAvidin Biotin Table 1. Spacer type Poly- or Oligo- Bioinert polymers Po|y(ethy|ene oxide/po|y(ethy|ene glycol), polypeptide, polyglycerol, polyoxazolines Nucleotide oligomers/polymers lbid Hydrocarbons Hexane, decane, pentadecane, octadecane, polyacetylene, polystyrene, polyethylene, Functional hydrocarbons Polyacrylamide, po|y(acry|ic acid), po|y(methy| methacrylate), Po|y(methy| acrylate) Table One or multiple linkers may facilitate one or multiple detection probes X and/or functional groups Y, or where multiple linkers can facilitate multiple detection probes and/or functional groups Y via an interconnecting backbone. For example a polymer chain with functional groups incorporated in the monomer such that multiple anchor and functional groups for linking to detection probe is incorporated in the chain, allowing the chain to orient and bind to the nanoparticle surface using its anchor groups and further be modified with detection probes using linkers that form bonds with the remaining functional groups.

Fluorophores One or more fluorophores may be selected from the alternatives provided in table 3 and deratives thereof.

Fluorophore Type Fluorophore Name Metallorganic Semiconductor Semiconductor quantum Dots (lll-V, ll-Vl, Si) Perovskite quantum dots Carbon quantum dots Quantum rods Blue Atto 425 4-[3~(etho> g]quinolšn-9(6H)~yi}butanošc acid Alexa fluortršslN,N-dEethylethanarramšuiïx) 8-[2-(4-{[(Zßvdioxopyrrolidin-l- yfioxykarborlyi}piperidira-LyEE-LoxoethaxylpyfreneáLšß-trisulforlate Green Alexa fluor6-amino-9-(2,4-dicarboxyphenyl)-4,5-disulfo-šH-xanthen-š-iminium Fluorescin 3',6'-dihydroxyspiro[isobenzofuran-1(3H),9'-[9H]xanthen]-3-one DiO 3,31Dioctadecyloxacarbocyanine Perchlorate AttoYellow - Orange Cy3 (amine and derivates) 6-[6-[(2E)-3,3-dimethyl-2-[(E)-3-(1,3,3-trimethy|indo|-1-ium-2-y|)prop-2- enylidene]indol-1-yl]hexanoylamino]hexylazanium;dichloride Dil 1,1'-Dioctadecyl-3,3,3',3'-Tetramethy|indocarbocyanine Alexa fluor2,3,5-trichIoro-4-{[({6-[(2,5-dioxopyrrolidin-1- yl)oxy]-6-oxohexy|}carbamoy|)methyl]su|fany|}-6- (2,2,4,8,10,10-hexamethyl-12,14-disu|fo- 2,3,4,8,9,10-hexahyd ro-1H-13-oxa-1, 11- diazapentacen-6-yl)benzoic acidAtto 550 N/A Red Cy5 6-[6-[(2E)-3,3-dimethyl-2-[(2E,4E)-5-(1,3,3-trimethy|indo|-1-ium-2- yl)penta-2,4-dienylidene]indol-1-yl]hexanoylamino]hexylazanium Alexa fluor 647 2-[5-[3,3-dimethyl-5-sulfo-1-(3-sulfopropy|)indo|-1-ium-2-yl]penta-2,4- dienylidene]-š-methyl-3-[5-oxo-5-(6-phosphonooxyhexylamino)penty|]- 1-(3-sulfopropyl)indo|e-5-sulfonic acid Texas Red 5-chlorosulfonyl-2-(3-oxa-23-aza-9- azoniaheptacyc|o[17.7.1.15,9.02,17.04,15.023,27.013,28]octacosa- 1(27),2(17),4,9(28),13,15,18-heptaen-16-yl)benzenesulfonate DiD 1,1'-Dioctadecyl-3,3,3',3'-Tetramethy|indodicarbocyanine Atto 647(N), Atto 655 N/A Near-IR Cyl-(E--carbfixypentyfi-Iâ--Ü-í l--ethyi -E--saašfo--lß--cšihycš ra-»Z H--indoi-»Z- y!Edene)hepta-l,Efié-»tršen-»l--yiLEH--indoišuan-ES--sušfønate Alexa fluor 680 N/A Alexa fluor 750 N/AAtto 680, AttO 700 N/A Table Nanoparticle The nanoparticle should fulfil the following Characteristics: - The size ofthe nanoparticle must be small enough for the nanoparticle to be able to penetrate the cell membrane, i.e. typically a diameter of less than 100 nm is required.

- The coating of the nanoparticle must allow specific targeting of the nanoparticle to the detection target in question. Also, it is important that the coating is such that aggregation is avoided, by tailoring the repulsive forces of the coating and/or modifying the formulation of the buffers used during binding to detection targets.

- The plexing capacity of the nanoparticle is important. Preferably more than 50 and up to or more than 100 different codes should be possible to detect. Thus, challenges of quenching, dye stability and level discernment must be solved. By carefully designing the nanoparticle architecture, optionally utilizing the size to further incorporate both organic dyes as well as inorganic dyes, and with the possibility to further modify the matrix and structure of the nanoparticle the stability of dyes can be increased as well as increasing the flexibility of combining various fluorophores that can work in synchronization to generate a high magnitude of optical encoding.

- Guidance for syntethization of nanoparticles for use according to the invention, with a suitable size, coating and fluorophore content, can be found in literature ofthe art, such as e.g. Wang et al. (Nanoletters, 2005; 5:1 (37-43)).

Target biomolecule preparation ln step c, the target biomolecule may be prepared by binding it to a molecule the method comprises the detection target, such as a barcoded padlock probe or initiator sequence for HCR or other amplification methods.

The detection target may be a nucleic acid molecule, which is amplified using a method as listed in tableMethod Details Rolling circle amplification mediated Padlock-probe mediated, clonal amplification method using the concept of rolling circle amplification. This is the most common in the emerging in-situ technologies.

Multiple hybridizations (ISH) mediated: HCR The concept of multiple hybridizations to (Hairpin chain reaction), and probably more generate a stronger signal is the common factor, one can design this in a multitude of different ways where the detection target is being built up using several hybridization events. This is what's been used traditionally with FISH.

Table Optical encoding & decoding The optical encoding may be effected by incorporating fluorophores of different wavelengths in precisely controlled ratios (intensities) such that each combination of wavelengths/intensity ratios becomes uniquely separable when decoding thereby optically encoding each nanoparticle type. The different ratios of intensities can be achieved by (i) controlling the concentration of fluorophores in the nanoparticle and/or (ii) altering the size or optical properties of the fluorophores to control its emission intensity and thereby achieving ratiometric read-out. lt is understood that the fluorophores incorporated in the nanoparticle may be encapsulated by its matrix and also placed at the surface ofthe nanoparticle.

Furthermore, each nanoparticle type is given a unique binding affinity through its coating such that it can recognize a unique detection target.

The decoding may be effected by optical decoding such as by optical imaging of the fluorescent signals. The signal from each nanoparticle type may be identified by measuring the wavelengths and intensities in order to ratiometrically identify the optical code incorporated in the nanoparticle types as shown in figure The target biomolecule may be further co-labelled with small molecule probes (comprising their own fluorophores) together with the nanoparticles. This increases the robustness of the assay by introducing a control signal showing where specific binding occurs between nanoparticles and detection target of the biomo|ecu|e(s), and furthermore may introduce another dimension in the optical encoding increasing the degree of multiplexity by combining the signal emitted from the nanoparticle with the signal emitted from the biomolecule. Therefore, the identity of the biomolecule can be determined by using optically encoded nanoparticles together with additionally encoding the biomolecule with non-nanoparticle probes, for example by extending the number of wavelengths (fluorophore signals) emitted by the nanoparticle-biomolecule complex by co-labeling with a probe containing a molecular fluorophore.

The person skilled in the art realizes that the present disclosure is not limited to the preferred embodiments described above. The person skilled in the art further realizes that modifications and variations are possible within the scope of the appended claims. Additionally, variations to the disclosed embodiments can be understood and effected by the skilled person in practicing the claimed disclosure, from a study of the drawings, the disclosure, and the appended claims.

EXAMPLES List of abbreviations: NPs: Nanoparticles EtOH: Ethanol PBS: Phosphate buffered saline DEPC: Diethyl pyrocarbonate treated RCA: Rolling circle amplification RCP: Rolling circle product, the result of rolling circle amplification BSA: Bovine serum albumin dNTP: Deoxyribonucleotide triphosphate ATP: Adenosine triphosphate PEG: Polyethylene glycol SSC: Saline-sodium citrate EDTA: Ethylenediaminetetraacetic acidExample 1 - Suspension conditions ln order to perform the claimed method, the conditions of the suspension, in which the method takes place, should have the following components and Characteristics: The suspension conditions must be controlled such that specific binding of nanoparticles to 5 detection targets is facilitated over unspecific binding to other targets or non-targets (such as other biological matter/matrix or surface) while maintaining the stability of nanoparticle dispersion.

The following components play important role in optimizing NP binding to target biomolecules Type (effect) Examples Range Denaturants (lower hybridization T) Formamide/Urea/ethylene glycol/ethylene carbonate/sodium percholarate etc. 0 - 50% (v/v) nonspecific binding) N-lauroylsa rcosine etc.

Salts & Buffer (lower NaCl 0 - 1000 mM electrostatic repulsion) Citrate buffer 0 - 100 mM Phosphate buffer 0 - 100 mM (Buffers usually used as SSC, PBS) etc. pH (charge of nucleic acids) Adjusted with HCl/NaOH 6 - 9 Nuclease inhibitor EDTA 0 - 10 mM Detergent (prevent SDS, Tween, 0 - 5% (v/v or m/v) Blocking agents (prevent nonspecific binding) BSA, denatured ssDNA etc. 0 - 10% (v/v or m/v) Hybridization accelerators dextran sulfate, PEG, ficoll STC. 0 - 20% (v/v or m/v) Table 5. Overview of components in probing/hybridization buffer. ln one embodiment, the following conditions were used:SSC 2X Formamide 20% PEG 4000 5% pH 7-7.ln another embodiment, the following conditions were used: SSC 1X Formamide 10% PEG 4000 5% pH 7-7.Example 2 - using the method for multiplexed detection ID Sequence 504013 TTTTTTTTTTCCTCAGTÄX/ÄTILÄGTGTCTTAC TTACACCCTTTCTTTGACAA GTGTATG CAGCTCCTCAGTAATAGTGTCTT 503525 TGTGCGTCTATTTAGTGGAGCC CATG GACTGTTACTGAGCTGCGTT 503344 TTGTCAAAGAAAGGGTGTAAAACGCAGCTCAGTAACAGTC 503793 TGCGTCTATTTAGTGGAGCC Table 6. Overview of sequences used in examples ln one embodiment, NP surface coating was performed by adding 100 pL of NPs (125 mM Si, 0.62 uM Cy3 loading) to a solution of H20 (485 pL) followed by L2-Azidosilane (2.5 pL, 256 mM), L1-mPEG5k Silane (10 pL, 20 mM) and finally ammonium hydroxide (2 pL, 28-30%). The temperature was raised to 75 °C and the reaction mixture was shaken. After 3 hours the reaction mixture was washed by centrifugation 3 times with H20 and 1 time with PBS. The coated NPs were stored in 100 pL H To EtOH (50 pL), above coated NPs were added (30 pL) followed by PBS (20 pL) and DBCO modified nucleotide sequence S04018 (3 pL, 100 uM). The temperature was raised to 37 °C 23 during shaking. After 19 hours the reaction mixture was washed by centrifugation 3 times with H20. The sequence functionalized NPs were stored in 100 pL H ln one embodiment, RCPs were prepared in solution by adding 5'-phosphory|ated padlock probes S03625 (1 pL, 1 uM) to a solution of H20 (71.9 uL), phi29 reaction buffer (10 uL, 10X), T4 ligase (0,4 pL, 5 U/pL), BSA (10 uL, 2 pg/pL), ATP (2.7 pL, 25 mM) followed by target sequence S03844 (4 uL, 30 nI\/|). The temperature was raised to 37 °C for 30 minutes. From this, 20 pL (first di|uted 111000) was added to H20 (9.2 uL) followed by BSA (4 uL, 2 pg/pL), dNTPs (2 pL, 2.5 mM), phi 29 reaction buffer (4 pL, 10X) and phi 29 polymerase (0,8 uL,U/pL). Temperature was raised to 30 °C for 2 hours followed by 65 °C for 5 minutes. ln one embodiment, NPs binding to RCPs were performed by first immobilizing 5 pL of RCPs to Superfrost Plus slides (Thermo Scientific) by deposition. The proceeding reactions were performed in Secure-seals (Grace Bio-Labs, 9 mm in diameter and 0.8 mm deep) attached to the slides. Hybridization mixture was prepared by adding NPs (5 pL) to a solution of SSC (5 pL, 20X), formamide (10 uL), H20 (24.5 pL), PEG 4000 (5 pL, 50%) followed by complementary detection oligo S03798-Cy5 (control probe). To the immobilized RCPs was added the hybridization mixture and temperature was raised to 37 °C. After one hour the glass slide was washed 4 times with PBS-T (DEPC-PBS with 0.05% Tween-20), once with 70% EtOH, once with 85% EtOH and finally 99.5% EtOH. ln one embodiment, RCP generation in situ was performed instead of in solution. Cells were seeded on Superfrost Plus slides (Thermo Scientific). When the cells reached the desired confluency they were fixed in paraformaldehyde (3% (w/v)) in PBS at room temperature. After 30 min, slides were washed twice with DEPC-PBS once with 70% EtOH, once with 85% EtOH and finally 99.5% EtOH for 3 min each. The reactions were performed in Secure-seals (Grace Bio-Labs, 9 mm in diameter and 0.8 mm deep) attached to the slides. To make the RNA more readily available for cDNA synthesis, 0.1 M HCI was applied to the cells for 10 min at room temperature. This was followed by two washes in DEPC-PBS.

Reverse transcription was performed by adding a mixture of DEPC-H20 (34.75 uL), TranscriptMe buffer (5 pL, 10X), dNTPs (1 pL, 25 mM), BSA (0.5 pL, 20 pg/pL), random decamers (2.5 uL, 100 uI\/|), RiboLock RNase lnhibitor (Fermentas) (1.25 pL, 40 U/pL) and reverse transcriptase (5 uL, 200 U/pL) to the cells and temperature was raised to 37 °C. After 24 18 hours, the reaction mixture was removed and paraformaldehyde (3% (w/v)) was added.

After 30 minutes the cells were washed with PBS-T twice.

Ligation was performed by adding a mixture of DEPC-H20 (22 uL), Ampligase buffer (20 mM Tris-HCI, pH 8.3, 25 ml\/l KCI, 10 ml\/l l\/lgC|2, 0.5 ml\/l NAD and 0.01% Triton X-100) (5 uL, 10X), KCI (2.5 uL, 1 M), formamide (10 uL), padlock probes (1 uL, 0.5 uI\/|), BSA (0.5 uL, 20 ug/uL), Ampligase (5 uL, 5 U/uL) and RNase H (4 uL, 5 U/uL) to the cells and temperature raised to°C for 30 minutes and then 45 °C. After 1 hour, cells were washed with PBS-T twice.

RCA was performed by adding a mixture of DEPC-H2o (33 uL), Phi-29 buffer (5 uL, 10X), glycerol (5 uL, 50%), dNTPs (0.5 uL, 25 mM), BSA (0.5 uL, 20 ug/uL), Exonuclease I (1 uL, 20 U/uL), Phi-29 polymerase (5 uL, 10 U/uL) to the cells and raising the temperature to 37 °C. After 4 hours, cells were washed with PBS-T twice. Subsequent NP and control probe binding was performed as above analogous to RCPs deposited on slides. ln one embodiment, image acquisition was performed using an Axioplan ll epifluorescence microscope (Zeiss) equipped with a 100 W mercury lamp, a CCD camera (C4742-95, Hamamatsu), and a computer-controlled filter wheel with excitation and emission filters for visualization of 425, DAPI, FITC, Cy3, Cy3.5, Cy5 and Cy7. A ><20 (Plan-Apocromat, Zeiss), ><40 (Plan- Neofluar, Zeiss) or ><63 (Plan-Neofluar, Zeiss) objective was used for capturing the images.

Claims (13)

1. Claims

2. A method for multiplexed detection of a plurality of target biomolecules in situ using optical encoding, thereby incorporating a combination of multiple wavelengths and intensities of fluorophores, wherein each target biomolecule has at least one detection target, comprising the steps of: a. providing a plurality of nanoparticle types in a suspension, each nanoparticle having a coating that provides binding affinity ofthe nanoparticle to a type- specific detection target, and wherein each nanoparticle comprises a plurality of fluorophores, that generates a signal which is unique for each nanoparticle type; b. providing one or more cells to be penetrated by the nanoparticles, wherein the ce||(s) comprise(s) a plurality of target biomolecules; c. optionally preparing the target biomolecules for binding with the nanoparticles, such as binding each target biomolecule with at least one molecule comprising the at least one detection target, and/or amplifying the detection target(s) in situ; d. contacting the cells with the suspension comprising the nanoparticles, thereby allowing the nanoparticles to penetrate the cells in order to bind with the detection targets of the target biomolecules to generate a ratiometrically identifiable signal; e. optically decoding the fluorophore signal(s) emitted by the nanoparticles of the nanoparticle type bound to the detection target of each target biomolecule by measuring the wavelengths and intensities of the emitted signals, thereby detecting the presence and identity of the target biomolecules.

3. The method according to claim 1, wherein each nanoparticle type is optically encoded by (i) incorporating precisely controlled ratios of the plurality of fluorophores, thereby controlling the emission wavelength and intensity from the nanoparticle type, or (ii) altering the properties of the fluorophores affecting its emission intensity.

4. The method according to claim 1 or 2, wherein the binding affinity of the coating of the nanoparticle type is provided by a detection probe X attached via a linker to the nanoparticle, wherein the detection probe X is chosen from a nucleic acid molecule, an antigen or an antibody.The method according to any of claims 1-3, wherein the coating of the nanoparticle furthermore comprises a repulsive component provided by a functional group Y attached via a linker to the nanoparticle, wherein the functional group Y is chosen from a charged group with a positive or negative charge, a zwitterionic group comprising both a positive or negative charge, or a sterically repulsive functional group such as polymer chain or an aliphatic chain.

5. The method according to any of claims 3 to 4, wherein the linker comprises at least one anchor group, which tethers the coating to the nanoparticle and optionally a spacer gfOLl

6. The method according to any one of claims 3 to 5, wherein one or more linkers can provide one or more detection probes X and/or functional groups Y, or where multiple linkers can provide multiple detection probes X and/or functional groups Y via an interconnecting backbone.

7. The method according to any one of claims 1 to 6, wherein the fluorophores are chosen from: (i) organic fluorophores, chosen from Atto 425, Cy3, Cy5, and Cy7; (ii) different colors of inorganic fluorophores, wherein the inorganic fluorophores comprises quantum dots, rods, perovskite quantum dots or metal-ligand complexes; and/or (iii) a combination of different colors of organic fluorophores and different colors of inorganic fluorophores, wherein the inorganic fluorophores comprises quantum dots, rods or perovskite quantum dots.

8. The method according to any one of claims 1 to 7, wherein the nanoparticle has at least one of the following Characteristics: i. it is a spherical particle comprising of silica and/or semiconductor, organic, inorganic, metal and/or polymer material; ii. it has a diameter of less than 300 nm, preferably less than 200 nm, and more preferably less than 100 nm, even more preferably less than 50 nm.

9. The method according to any one of claims 1 to 8, wherein in step c, target biomolecule is prepared by binding to it at least one molecule comprising the detection target, such as a barcoded nucleic acid molecule, padlock probe or initiator sequence for subsequent amplification.

10. The method according to any one of claims 1-9, wherein the detection target comprises a nucleic acid molecule, which is, or facilitates a molecule that is, amplified using RCA or multiple hybridization events.

11. The method according to any one of claims 1 to 10, wherein the decoding is effected by optical decoding such as by optical imaging.

12. The method according to any one of claims 1-11, further providing one or more molecular probes, wherein each molecular probe comprises a fluorophore that is bound to a nucleic acid molecule, an antigen or an antibody providing binding affinity ofthe molecular probe to the specific detection target.

13. The method according to any one of claims 1-12, wherein the nanoparticles are provided in a kit of parts; the kit of parts comprising, in separate containers, (i) nanoparticle(s) of one or more types in a suspension, each nanoparticle type having a coating that provides binding affinity of the nanoparticle to a specific detection target, and wherein each nanoparticle comprises a plurality of fluorophores that generates a signal which is unique for each nanoparticle type, optionally nanoparticle(s) of each type in a suspension, each nanoparticle type having a coating without binding affinity of the nanoparticle to a specific detection target wherein the binding affinity is provided in a subsequent step by a detection probe X attached via a linker to the nanoparticle, wherein the detection probe X is chosen from a nucleic acid molecule, an antigen or an antibody; (ii) optionally, ingredients for providing the one or more nanoparticle type(s) with binding affinity to a specific detection target, comprising a reaction buffer facilitating thebinding of detection probe X to the linker, a washing buffer, and a suspension buffer to suspend the nanoparticles in after the introduction of the binding affinity to the coating; (iii) a probing buffer, comprising a solution with controlled pH, salt concentration and additives facilitating specific detection target binding of the nanopartic|e(s); and (iv) instructions for use of the kit in the method according to claim 1-12.